pET vector cloning of hydrophobic protein gene
hdang at channel.neusc.bcm.tmc.edu
Wed Aug 2 14:26:28 EST 1995
> In article <3vltmi$qn5 at warp.cris.com>, mike.holloway at stjude.org (Mike
> Holloway) wrote:
> > I'm giving up on the pET system and wondered if anyone else has had
> > similar experiences. Even with the thioredoxin fusion protein vector,
> > I can not isolate a transformant with my sequence and an intact vector.
> > I do get many rearrangements - things that are not whole pET vector or
> > PCR template from the preparation of my insert. The transformants are of
> > various sizes, with various restriction sites missing. Other cloning
> > projects using the same bugs and reagents are working just fine, without
> > any evidence of contamination.
> > I have to conclude that some protein is being made, despite what anyone
> > says about the T7 promoter not being used without the polymerase being
> > pumped out, and that its toxic. This has been the previous experience
> > in our lab with this protein using other bacterial expression vectors.
> > Apparently this isn't an isolated experience, since several companies
> > have recently come out with this thioredoxin fusion protein scheme that's
> > supposed to solve everything.
> > Anyone had experience with pET vectors?
> > =========================================================================
There may be 1 way out: recently people in our lab has succeeded in
cloning one of
those "toxic" thing in a pET, which had failed before.
Here's the trick: a PCR product was direction-cloned into both pET-20b and
1st one, no colony, 2nd one, many. The relevant difference, there is a
T7 promoter in 25b. So in addition to controlling T7 pol, 25b also has a
on your gene of interest that may further cut down on leaky expression of
and make it tolerable for the bug.
So seems there is a dose effect, if your protein is really, really toxic
to the bug, it
still may not express. But try the T7-lac combo vector before giving it
up, since you
might hit the same problem with another system as well.
Hong Dang (hdang at channel.neusc.bcm.tmc.edu)
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